RESUMO
Nipah virus (NiV) is a recently emerged zoonotic paramyxovirus whose natural reservoirs are several species of Pteropus fruit bats. NiV provokes a widespread vasculitis often associated with severe encephalitis, with up to 75% mortality in humans. We have analyzed the pathogenesis of NiV infection, using human leukocyte cultures and the hamster animal model, which closely reproduces human NiV infection. We report that human lymphocytes and monocytes are not permissive for NiV and a low level of virus replication is detected only in dendritic cells. Interestingly, despite the absence of infection, lymphocytes could efficiently bind NiV and transfer infection to endothelial and Vero cells. This lymphocyte-mediated transinfection was inhibited after proteolytic digestion and neutralization by NiV-specific antibodies, suggesting that cells could transfer infectious virus to other permissive cells without the requirement for NiV internalization. In NiV-infected hamsters, leukocytes captured and carried NiV after intraperitoneal infection without themselves being productively infected. Such NiV-loaded mononuclear leukocytes transfer lethal NiV infection into naïve animals, demonstrating efficient virus transinfection in vivo. Altogether, these results reveal a remarkable capacity of NiV to hijack leukocytes as vehicles to transinfect host cells and spread the virus throughout the organism. This mode of virus transmission represents a rapid and potent method of NiV dissemination, which may contribute to its high pathogenicity.
Assuntos
Leucócitos/virologia , Vírus Nipah/fisiologia , Animais , Sequência de Bases , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Primers do DNA , Humanos , Mesocricetus , RNA Viral/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Vero , Replicação ViralRESUMO
Free-living amoebae are known to act as replication niches for the pathogenic bacterium Legionella pneumophila in freshwater environments. However, we previously reported that some strains of the Willaertia magna species are more resistant to L. pneumophila infection and differ in their ability to support its growth. From this observation, we hypothesize that L. pneumophila growth in environment could be partly dependent on the composition of amoebic populations and on the possible interactions between different amoebic species. We tested this hypothesis by studying the growth of L. pneumophila and of a permissive free-living amoeba, Vermamoeba vermiformis (formerly named Hartmannella vermiformis), in co-culture with or without other free-living amoebae (Acanthamoeba castellanii and W. magna). We demonstrate the occurrence of inter-amoebic phagocytosis with A. castellanii and W. magna being able to ingest V. vermiformis infected or not infected with L. pneumophila. We also found that L. pneumophila growth is strongly impacted by the permissiveness of each interactive amoeba demonstrating that L. pneumophila proliferation and spread are controlled, at least in part, by inter-amoebic interactions.
Assuntos
Amébidos/microbiologia , Legionella pneumophila/crescimento & desenvolvimento , Fagocitose , Amébidos/classificação , Amébidos/crescimento & desenvolvimento , Técnicas de Cocultura , Interações entre Hospedeiro e Microrganismos , Doença dos Legionários/transmissão , Microbiologia da ÁguaRESUMO
We previously demonstrated the importance of upregulation of phosphatidylethanolamine N-methylation pathway in euryhaline fish and crustaceans facing hyperosmotic conditions. In marine molluscs phosphatidylcholine synthesis through N-methylation of phosphatidylethanolamine has not been described until now. In vivo labeling of the mussel Mytilus galloprovincialis with [1-(3)H]-ethanolamine showed that the digestive gland is the tissue expressing the highest incorporation into lipids. A sustained increase in lipid labeling was observed up to 72 h following label injection with 79-92% of radioactivity concentrated into phosphatidylethanolamine and phosphatidylcholine. A direct correlation (r = 0.47, p < 0.01) between the specific radioactivities of phosphatidylcholine in plasma and the digestive gland was observed. Moreover, the phosphatidylcholine fatty acid compositions of plasma and the digestive gland were similar but differed from those of phosphatidylcholine purified from other tissues. In vitro incubation of tissues with [1-(3)H]-ethanolamine or L-[3-(3)H]-serine showed that a significant labeling of the choline moiety of phosphatidylcholine was observed in the digestive gland and hemocytes. Pulse-chase experiments with [1-(3)H]-ethanolamine also demonstrated that hemocytes are exchanging the newly formed phospholipids with plasma. Finally, phosphatidylethanolamine N-methyltransferase assays demonstrated salinity-dependent activities in the digestive gland and hemocytes. We conclude that in M. galloprovincialis an active phosphatidylcholine synthesis through N-methylation of phosphatidylethanolamine occurs in the digestive gland and hemocytes and that this newly formed phosphatidylcholine is partly exchanged with plasma.
Assuntos
Organismos Aquáticos/fisiologia , Trato Gastrointestinal/metabolismo , Hemócitos/metabolismo , Mytilus/fisiologia , Fosfatidilcolinas/biossíntese , Fosfatidiletanolaminas/metabolismo , Animais , Etanolamina/metabolismo , Hidrogênio/metabolismo , Metilação , Concentração Osmolar , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Radioisótopos/metabolismo , Água do Mar , Serina/metabolismoRESUMO
Phosphatidylcholine (PC), the main phospholipid in eukaryotes, is synthesized via two different routes, the phosphatidylethanolamine N-methyl transferase (PEMT) and the CDP-choline pathways. We previously showed in euryhaline fish that salinity impacts the relative contribution of the two pathways for PC biosynthesis, with PEMT pathway being activated in the liver of sea water (SW)-adapted animals. To address the occurrence of such phenomenon in other animals we performed in vivo metabolic studies in two crustacean species: the Chinese crab (Eriocheir sinensis) and the green crab (Carcinus maenas). In both species, the levels of PC and phosphatidylethanolamine in hepatopancreas and hemolymph were not modified by SW-adaptation. In E. sinensis, SW-adaptation activated PC labeling from L-(U-(14)C)-serine in the hepatopancreas and resulted in an increased ratio of PC specific activities between hemolymph and hepatopancreas. In C. maenas, incorporation of L-(3-(3)H)-serine and L-(2-(14)C)-ethanolamine into PC of hepatopancreas was strongly inhibited after acclimation to fresh water (FW). The results show that PC synthesis via the PEMT pathway and its subsequent release into hemolymph are both activated in SW- compared to FW-adapted animals. SW-adaptation also resulted in increased tissue concentrations of betaine and labeling from L-(U-(14)C)-serine, suggesting that the PEMT-derived PC is used for the synthesis of organic osmolytes. The physiological relevance of these observations is discussed.
Assuntos
Crustáceos/metabolismo , Hemolinfa/metabolismo , Hepatopâncreas/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Salinidade , Adaptação Fisiológica , Animais , Metilação , Fosfatidilcolinas/biossínteseRESUMO
PURPOSE: We initiated studies on the mechanisms of cell death in head and neck squamous cell carcinoma cell lines (HNSCC) since recent clinical trials have shown that local treatment of HNSCC by carbon hadrontherapy is less efficient than it is in other radioresistant cancers. METHODS AND MATERIALS: Two p53-mutated HNSCC cell lines displaying opposite radiosensitivity were used. Different types of cell death were determined after exposure to carbon ions (33.6 and 184 keV/microm) or X-rays. RESULTS: Exposure to radiation with high linear energy transfer (LET) induced clonogenic cell death for SCC61 (radiosensitive) and SQ20B (radioresistant) cells, the latter systematically showing less sensitivity. Activation of an early p53-independent apoptotic process occurred in SCC61 cells after both types of irradiation, which increased with time, dose and LET. In contrast, SQ20B cells underwent G2/M arrest associated with Chk1 activation and Cdc2 phosphorylation. This inhibition was transient after X-rays, compared with a more prolonged and LET-dependent accumulation after carbon irradiation. After release, a LET-dependent increase of polyploid and multinucleated cells, both typical signs of mitotic catastrophe, was identified. However, a subpopulation of SQ20B cells was able to escape mitotic catastrophe and continue to proliferate. CONCLUSIONS: High LET irradiation induced distinct types of cell death in HNSCC cell lines and showed an increased effectiveness compared with X-rays. However, the reproliferation of SQ20B may explain the potential locoregional recurrence observed among some HNSCC patients treated by hadrontherapy. An adjuvant treatment forcing the tumor cells to enter apoptosis may therefore be necessary to improve the outcome of radiotherapy.
Assuntos
Carcinoma de Células Escamosas/radioterapia , Morte Celular , Neoplasias de Cabeça e Pescoço/radioterapia , Tolerância a Radiação , Proteína Quinase CDC2/metabolismo , Radioisótopos de Carbono/farmacologia , Morte Celular/fisiologia , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Fase G2/efeitos da radiação , Genes p53/genética , Humanos , Transferência Linear de Energia , Mitose/efeitos da radiação , Proteínas Quinases/metabolismo , Tolerância a Radiação/genética , beta-Galactosidase/metabolismoRESUMO
Gaucher disease is an inherited metabolic disorder caused by defective activity of the lysosomal enzyme, glucocerebrosidase, resulting in accumulation of the lipids, glucosylceramide (GlcCer), and glucosylsphingosine (GlcSph). Little is known about the mechanism leading from lipid accumulation to disease, particularly in the acute and subacute neuronopathic forms of Gaucher disease, types 2 and 3, respectively. Recent work from our laboratory has shown, in animal models, that GlcCer enhances agonist-induced calcium release from intracellular stores via the ryanodine receptor, which results in neuronal cell death. We now test whether calcium release is altered in human brain tissue obtained post-mortem from Gaucher disease patients. Agonist-induced calcium release via the ryanodine receptor was significantly enhanced (P < 0.05) in brain microsomes from the acute neuronopathic form of Gaucher disease (type 2) (43 +/- 6% of the calcium in microsomes) compared to the subacute (type 3) (27 +/- 3%) and the non-neuronopathic (type 1) (28 +/- 6%) forms, and controls (18 +/- 3%), and correlated with levels of GlcCer accumulation. These findings suggest that defective calcium homeostasis may be a mechanism responsible for neuropathophysiology in acute neuronopathic Gaucher disease, and may potentially offer new therapeutic approaches for disease management.
Assuntos
Encéfalo/metabolismo , Sinalização do Cálcio/genética , Cálcio/metabolismo , Doença de Gaucher/metabolismo , Regulação para Cima/genética , Doença Aguda , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Encéfalo/patologia , Encéfalo/fisiopatologia , Química Encefálica , Agonistas dos Canais de Cálcio/farmacologia , Pré-Escolar , Doença de Gaucher/patologia , Doença de Gaucher/fisiopatologia , Glucosilceramidas/metabolismo , Humanos , Lactente , Recém-Nascido , Microssomos/química , Microssomos/metabolismo , Pessoa de Meia-Idade , Canal de Liberação de Cálcio do Receptor de Rianodina/genéticaRESUMO
Gaucher disease is caused by defective activity of acid-beta-glucosidase (GlcCerase), resulting in accumulation of glucosylceramide (GlcCer) mainly in macrophages. We now demonstrate that secondary biochemical pathways regulating levels of phospholipid metabolism are altered in a Gaucher disease macrophage model. Upon treatment of macrophages with the GlcCerase inhibitor, conduritol-B-epoxide, phosphatidylcholine (PC) labeling with the metabolic precursor, [methyl-14C]choline, was elevated after 6 or 12 days in macrophages but not in lymphocytes. These changes correlated with increases in the cytoplasmic/nuclear ratio and with levels of [3H]GlcCer accumulation. Moreover, metabolic labeling with L-[3-3H]serine and L-[methyl-3H]methionine demonstrated that PC synthesis via the methylation of phosphatidylethanolamine is also increased in CBE-treated macrophages. Since PC is a major structural component of biological membranes and the source of various second messengers, we suggest that changes in its metabolism in macrophages may be relevant for understanding Gaucher disease pathology.
Assuntos
Doença de Gaucher/metabolismo , Inositol/análogos & derivados , Linfócitos/metabolismo , Macrófagos/metabolismo , Fosfatidilcolinas/biossíntese , Fosfatidilcolinas/metabolismo , Isótopos de Carbono , Núcleo Celular/química , Citoplasma/química , Deutério , Glucosilceramidase/antagonistas & inibidores , Glucosilceramidas/metabolismo , Humanos , Inositol/farmacologia , Macrófagos/patologia , Modelos Biológicos , Monócitos/citologia , Fosfatidilcolinas/análise , Fosfatidiletanolaminas/metabolismo , Regulação para CimaRESUMO
We describe a new method that permits quantification in the pmol to nmol range of three lyso-neutral glycosphingolipids (lyso-n-GSLs), glucosylsphingosine (GlcSph), galactosylsphingosine (GalSph), and lactosylsphingosine, in the same sample as neutral glycosphingolipids (n-GSLs). Lyso-n-GSLs and n-GSLs are initially obtained from a crude lipid extract using Sephadex G25 chromatography, followed by their isolation in one fraction, which is devoid of other contaminating lipids, by aminopropyl solid-phase chromatography. Lyso-n-GSLs and n-GSLs are subsequently separated from one another by weak cation exchange chromatography. N-GSLs are then deacylated by strong alkaline hydrolysis, and the N-deacylated-GSLs and lyso-n-GSLs are subsequently N-acetylated using [3H]acetic anhydride. An optimal concentration of 5 mM acetic anhydride was established, which gave >95% N-acetylation. We demonstrate the usefulness of this technique by showing an approximately 40-fold increase of both GlcSph and glucosylceramide in brain tissue from a glucocerebrosidase-deficient mouse, as well as significant lactosylceramide accumulation. The application and optimization of this technique for lyso-n-GSLs and lyso-GSLs will permit their quantification in small amounts of biological tissues, particularly in the GSL storage diseases, such as Gaucher and Krabbe's disease, in which GlcSph and GalSph, respectively, accumulate.